This proposal concerns a biochemical investigation of mechanisms involved in the transcription of specific genes during normal cell growth and differentiation and in malignant cells. The major emphasis will be on specific 5S RNA, tRNA and histone genes expressed in Xenopus laevis (amphibian) oocytes and embryos, in mouse plasmacytoma cells, or in Drosophila melanogaster. The general approach involves an analysis of the in vitro transcription of these genes by homologous class I, II, or III RNA polymerases. Recent studies in this laboratory have demonstrated the selective and accurate transcription of specific genes (both cellular and viral) in chromatin, but not in purified DNA, by purified class III RNA polymerases. Thus, further attempts to elucidate specific transcriptional regulatory elements will involve analysis of (1) the function of specific RNA polymerase components (subunits or associated factors) as well as specific modifications thereof (e.g. phosphorylation). (2) Isolation and characterization of those components associated with actively transcribed genes, including an analysis of the structure of the actively transcribed genes. (3) Reconstruction from purified components (both proteins and specific gene fragments) of simple well defined templates which are accurately transcribed by the appropriate RNA polymerase and by other necessary (e.g. soluble) cellular factors. (4) Possible chromatin structural modifications (e.g. acetylation) during the process of gene activation or transcription. (5) Detailed studies of the interactions and functions of specific structural and regulatory proteins and nucleic acid sequences. (6) Comparative studies, in heterologous reconstructed systems, of the regulatory components for analogous genes from different cells types. In all cases the significance of specific transcription events will be ascertained by detailed comparisons of in vivo and in vitro transcripts and by correlations between in vitro functions and in vivo events. The technologies used will include: affinity chromatography, immunoadsorption, recombinant DNA cloning, nucleic acid hybridization and sequence analysis restriction endonuclease digestion, and chromatin fractionation, reconstitution and analysis.